Currently, there are many human diseases—such as inflammatory pathologies, autoimmune diseases, or acute pain—which have to be endured by people, which require development of new technologies to advance diagnosis or accelerate and improve therapeutic procedures which can overcome or at least alleviate a disease.
In spite of recent advances in biotechnology and current medicine associated to prevention, early diagnosis, and therapeutic treatments, there are still many challenges to improve the early diagnosis and the life quality related to therapies. Without going too far, and as an example, cancer is still a high mortality index disease which only in the United States takes more than 500,000 lives per year.
Given the need to improve the diagnostic and therapeutic systems, including the enormous advances that modern medicine has made, the development of new materials and their combination has been favored, allowing the transport and delivery of specific molecules, either probes in case of need of a diagnosis, or therapeutic compounds or drugs, proteins or nucleic acids, acting in very precise locations inside the animal or human body which is in need thereof.
For example, gene therapy advances considers the transport of isolated molecules of nucleic acids, such as genes and/or small interference RNA molecules (siRNA) to specific cells.
Exogenous siRNA can regulate the expression of genes codifying proteins associated with a particular pathology, as long as they are incorporated directly into the cells where specific gene expression regulation is required. Nevertheless, therapeutic applications of siRNA are precisely restricted due to unsolved problems involving efficient transport and delivery to the specific zone where their action is required: intracellular medium of those specific cells inside a person in need of a therapeutic treatment.
An efficient delivery of siRNA therefore requires improving incorporation thereof inside the cells to be treated, and previously, avoiding its degradation in blood and extracellular medium. Precisely, the present invention shows an improved alternative for transport and delivery of molecules, such as siRNA, using a new transport polymeric structure constituted by PAMAM (polyamidoamine) dendrimers, a spacer molecule, and cafestol.
Great part of the advances in the development of new transport and delivery materials for use with biocompatible active substances in the treatment of animals and humans in particular, are associated with the design of new compositions investigated in the field of nanotechnology, which are also useful as carrier vehicles for probing molecules or molecules with a therapeutic end, and which are able to recognize, with a high precision degree, target cells to which their valuable material of last generation molecules with biomedical activity, either diagnostic or therapeutic.
As previously mentioned, one of the advantages that must be assured in the development of new transport and delivery carriers for support substances in medical treatments, is that the carrier is conformed in base of polymers which can bind active molecules and transport them to a predetermined location. These polymers must have innocuous features, at systemic level as well as in a particular organ, tissue, or target cells where it is expected that the diagnostic or medical molecule is delivered, avoiding degradation of the transported molecule, and therefore assuring efficient distribution to the targeted location, mainly at intracellular level.
Thus, it is important to take into account that the polymeric structure of these vehicles or carriers must be characterized by showing a solubility compatible with the fluids with which will interact until arriving to its destination, in such a manner to improve its stability—and the transported molecule—during the journey and favoring delivery times to the target site of said active molecule to be distributed, especially when the latter shows a low solubility degree, that prevents arriving by itself to the place where its action is expected to occur.
In this way, the carrier materials must comply with the need of avoiding fast or systematic degradation of themselves as well as the transported molecule and during all the journey to the targeted site. It is also expected that once the active substance is delivered, the carrier polymers should be degraded or eliminated from the patient's body, as long as the whole process does not produce co-lateral damages.
All of the above can be summarized in that a good active molecule, with a biomedical utility, carrier system must favor arrival and accumulation of an intact active substance, either at organ level, in a tissue or to targeted cells. Among the last generation carriers which have been developed for in vivo transport of drugs, proteins or isolated nucleic acids, we can find polymeric micelles, hydrogels, liposomes, or water-soluble conjugated polymers, developed in great part by application laboratories in the field of nanotechnology.
As such, nanotechnology associated to medicine, is born through convergence and complementarity of scientific findings obtained from research in physical, chemical, and biological sciences, being considered nowadays as one of the most promising technologies in the field of human and animal health in general (Ann. Bioanal. Chem.: 400: 483-492). Among the technological advances made in the field of nanotechnology reported during the last five years, different polymeric approaches have been reported, related to structures in the scale between 1 and 100 nanometers (1-100 nm). Among the advances in nanotechnological structures platforms, we can consider four of them as widely acknowledged by favoring production of relatively precise nano-structures, such as fullerenes, nanotubes, quantum dots, and dendrimers.
A family of potential carriers for drug and/or gene delivery are dendrimers, which are polymers (macromolecules between 5,000 and 500,000 g/mol), mono-disperse, spherically shaped, well defined and regularly branched, and which can be easily modified or “activated” in their surfaces with functional moieties such as amines (—NH2), carboxylates (—COOH), etc., which allow coupling to said modified dendrimers of biomedical important substances which need to be transported and delivered to very specific locations (organs, tissues, or specific cells) inside an animal or a person.
Dendrimers are therefore, potential polymeric structures which can be used as carrier vehicles for the administration of either drugs, proteins, or isolated nucleic acids, associated to therapeutic procedures wherein the administration to a patient of active substances to be distributed to preferred tissues, at a controlled or preferred delivery rate, and protected from metabolic degradation before arrival to their final destination where its action is required, is needed. Also, the relatively empty intra-molecular cavity that dendrimers have, would facilitate the “molecule hosting”, providing new opportunities for further designs of delivery of drugs and genes.
Structurally, dendrimers are a class of macromolecules at a nanometric scale (less than 1/1,000,000 meters), and therefore they are also known as nanostructures, having multivalent surfaces allowing further modification with functional moieties (—NH2, —COOH, alkyl, etc.), facilitating later the binding of probing molecules for diagnosis or molecular agents for the treatment of diseases.
The chemical versatility of dendrimers has produced its intense research in the last years, due to its potential use in biomedical applications such as gene therapy, in vivo drug or therapeutic molecules or diagnosis probes delivery.
These nano-structures (dendrimers) have the versatility to allow conjugation at surface level of their polymeric structure, with different functional moieties useful in modern molecular medicine (probes, nucleic acids, proteins or medicinal drugs). Nevertheless, conjugation of said functional moieties to the dendrimer depends on the reactivity of the peripheral groups present in the dendrimer. Therefore, there is also the need to broaden the range of functional moieties that can be present in the surface of the dendrimer, and thus, increasing the possibility of conjugation of different and new alternative probe molecules or medicinal drugs, to the surface of the dendrimer, and which can be delivered to a target organ, tissue, cell, for those active substances.
Since the utility of dendrimers is widely recognized as a modern tool for transport of other active molecular agents, many types of dendrimers have been developed and employed in many applications including medicine, chemistry, and pharmacy.
This has produced the development of hundreds of polymeric drug and therapeutic molecular agents carriers, in many laboratories worldwide, being the ones based in PAMAM (PolyAMidoAMine) well-known as highly efficient in the transport and delivery of genes, as efficient as those based in polyethylenimine (PEI), recognized for being the most efficient carriers known nowadays.
Literature teaches many different approaches for preparing dendrimers which are functionalized for recognizing determined cell types, through conjugation with ligands that can interact with target molecules in specific tissues and/or defined cells (for example, through recognition and binding to surface membrane receptors of said target cells). Some background shown in the literature regarding conjugated polymers with potential application in biomedicine is mentioned here below.
Patent application WO2011053618 teaches new procedures for the synthesis of dendrimers based in PAMAM modified in its ends with multiple hydroxyl groups or ethylenglycol oligos, which are compatible with further conjugation with functional ligands such as therapeutic agents, drugs, probes, etc.
PAMAM dendrimers have been used widely in the design of new hydrogels formed as small cross-linked particles in a size ranging from 25 nm a 10 μm (10,000 nm) which are appropriate according to the inventors (check international patent application WO2011123591) for distributing hydrogel through any orifice, tissue, muscle route, subcutaneous or ocular way of the body of a patient in treatment or under observation for diagnosis. In the hydrogels of WO2011123591, PAMAM dendrimers are cross-linked with asymmetrical terminal groups in such a manner that a terminal group is directly involved in the formation of the hydrogel and the other is enabled to conjugate—and thus bind and transport—therapeutic or diagnostic molecules which are to be distributed with the hydrogel.
Among the applications in medicine which have been published using dendrimers, the use of PAMAM for treating inflammatory disorders, and those of autoimmune nature, such as rheumatoid arthritis, osteoarthritis, juvenile idiopathic arthritis, psoriasis, lupus erythematosus, Crohn's disease, or sarcoidosis, and where PAMAM dendrimers are used for the transport of therapeutic agents, for recognition or diagnosis associated to the mentioned diseases has been considered (check international patent application WO2010054321).
Patent application US2010158850 describes coupling of two dendrimers, preferentially PAMAM and POPAM (PolyPropylAMine) and wherein each of the coupled dendrimers is modified by functional groups allowing further conjugation with therapeutic agents, diagnostic therapeutic pre-treatment or for evaluation of results of a given therapy, for example against cancer, through direct recognition of surface receptors in target cells by these polymeric structures coupled with selected therapeutic or diagnostic agents.
Patent application WO2010147831 describes as a carrier for active agents, a composition comprising N-acetyl cysteine bound to PAMAM dendrimer, and wherein the latter also binds to the active agent to be transported, through disulfide bonds.
Notwithstanding the foregoing, the use of PAMAM as structural base in the development of carriers and delivery agents in situ and in vivo for genes, therapeutic drugs, or diagnostic probes, presents some problems which are to be solved and which are associated mainly to a low solubility of these dendrimers in water, as well as certain toxicity and low capacity in penetrating the cell membrane. This has led to research modifications to this PAMAM dendrimers, binding them to other polymers such as polyethylene glycol (PEG), since the latter shows very low immunogenicity, is biocompatible and presents high solubility in water (Kim et al (2004), Biomacromolecules 5: 2487-2492).
Among the polymers developed for improving the in vivo efficiency of these carriers for genes, therapeutic drugs and/or diagnostic probes, and combining the properties of PAMAM and PEG, the work of Kim et at (Biomacromolecules 5: 2487-2492, 2004) can be mentioned. They developed a hybrid, self-assembling co-polymer, and based in the order of a polymeric structure of three blocks: PAMAM-PEG-PAMAM. Said structure shows an advance over water solubility compared to polymers based exclusively in PAMAM, but still has cytotoxicity and transfection problems. Therefore, this cannot be referred as an improved carrier, and thus, it is not fit for use in biomedical treatments.
As summarized, notwithstanding the broad impulse currently given worldwide to the development of new generations of in vivo carrier polymers for medical support substances in therapy or diagnosis, the need of developing new polymeric preparations, which are not only biocompatible, with a good cellular permeability, and not toxic to the treated organism, but also showing higher versatility for coupling new generations of therapeutic or diagnostic substances still to be developed which will require precise transportation to a specific target in a patient in need thereof, still exists.